Preservation versus resection of Denonvilliers’ fascia in total mesorectal excision for male rectal cancer: follow-up analysis of the randomized PUF-01 trial

Traditional total mesorectal excision (TME) for rectal cancer requires partial resection of Denonvilliers’ fascia (DVF), which leads to injury of pelvic autonomic nerve and postoperative urogenital dysfunction. It is still unclear whether entire preservation of DVF has better urogenital function and comparable oncological outcomes. We conducted a randomized clinical trial to investigate the superiority of DVF preservation over resection (NCT02435758). A total of 262 eligible male patients were randomized to Laparoscopic TME with DVF preservation (L-DVF-P group) or resection procedures (L-DVF-R group), 242 of which completed the study, including 122 cases of L-DVF-P and 120 cases of L-DVF-R. The initial analysis of the primary outcomes of urogenital function has previously been reported. Here, the updated analysis and secondary outcomes including 3-year survival (OS), 3-year disease-free survival (DFS), and recurrence rate between the two groups are reported for the modified intention-to-treat analysis, revealing no significant difference. In conclusion, L-DVF-P reveals better postoperative urogenital function and comparable oncological outcomes for male rectal cancer patients.

Reviewer #1: Remarks to the Author: This is a well conducted randomized controlled trial comparing preservation or en bloc resection of Denonvilliers' fascia in male patients with rectal cancer that is not located anteriorly or confined to the rectal wall (max.T2) if located on the anterior side.None of the patients had preoperative radiotherapy.The results show better urogenital function if preserving Denonvilliers' fascia, which is also what I would have expected beforehand.The manuscript is of good textual quality.
The authors state that traditional TME requires partly resection of Denonvilliers' fascia, but in my view this is debatable and not generally acknowledged anymore.The rationale of performing this trial in the current era is not completely clear to me, because TME surgery is often individualized based on tumour location and extent since we have detailed information from preoperative MRI, aiming at an optimal balance between oncological safety and nerve preservation.Therefore, most surgeons will not resect Denonvilliers' fascia if the tumour is located on the dorsal or dorsolateral side, and this might even be safe if there is at least 2 mm margin on MRI to this fascia in anteriorly located tumours.The authors confirm that preserving Denonvilliers' fascia, if oncologically safe, gives the best outcomes.But in my opinion, this is common practice, at least in my country, and the trial does not provide any practice changing results.This discussion is especially relevant, since only a quarter of included patients had an anteriorly located tumour (Table 1).Therefore, the issue of preservation of Denonvilliers' fascia was only clinically relevant in a minor subgroup.
There is an important issue related to extrapolation of these results to other countries and patient populations.Western populations might be different due to obesity and different anatomy, and more application of neoadjuvant radiotherapy.
The two types of surgery are described as straight forward, but in reality it might sometimes be quite difficult to clearly identify Denonvilliers' fascia and to choose the plane either ventral or dorsal to this fascia.This is also due to high variability in appearance of Denonvilliers' fascia in different patients, as pointed out by the authors in the discussion.The authors describe video registration and photographs for quality control of the surgical interventions, but do not provide any results for assessment of video's or pictures.Some detailed comments: Title: it is not clear from the title that this is an RCT Discussion: This is too long and might benefit from more focus on the main findings of the study and comparing their results with urogenital function in other studies that included these outcomes, not necessarily looking at DVF preservation.The detailed anatomical discussion is probably something for a narrative review, but not as part of an original manuscript on an RCT.The authors state that it would be interesting to also look at anterior T3 and T4 tumours, but this seems impossible or even unethical, as DVF is at least threatened or even involved in those patients.Please explain.The fact that female patients were not included is not a limitation, because the DVF is not a clearly defined structure in women, and therefore the research question is not valid for this population.
Reviewer #2: Remarks to the Author: Inclusion of Denonvilliers' fascia became a standard in total mesorectal excision (TME) for rectal cancer.The primary aim was to improve the oncological outcome in the area with the open surgical approach.With the laparoscopic approach the visibility of the anatomic structures has improved with the possibilities of more accurate dissection that may preserve the Denonvilliers' fascia and reducing the risk of traumatizing the neurogenetic innervation of the bladder and sexual function in men.
In this elegant and very well-designed randomized study the results showed that the preservation of Denonvilliers' fascia significantly improved the sexual and urinary functional outcome without compromising the oncological outcome.This indeed is new and important knowledge with a high impact on the surgical treatment of rectal cancer in men.Inclusion and exclusion criteria are welldescribed and relevant.A strength is that those health care professionals that investigated the primary outcomes were blinded to the intervention performed.Thus, the results are very reliable.The statistical methods are appropriate.Another a strength is that professor Heald (the "father of TME") has been a member of the international advisor group in this study.The manuscript is very well-written.
The only thing a mis is information how many patients that were included at each participating center and whether all patients planned for a low anterior resection for curative intention were evaluated and included in the "group of eligible patients".
Reviewer #3: Remarks to the Author: This paper presents the analysis of secondary outcomes (OS and DFS) of a randomized clinical trial evaluating DVF preservation versus resection.The primary analysis regarding the effect on postoperative urogenital function was previously published.There are several flaws in the analysis and inconsistencies in the presented results that are of major concern and limit the interpretation of the results as presented.These are listed in detailed below.Major concerns: 1.The results in the text do not match the survival graphs and eTable5 presented.This is of major concern.The survival graphs sample size also don't match Table 1.For example, the log-rank pvalues are different and the sample sizes are different for the stratified analysis.2. The analysis is stated as intention to treat, but it is per protocol.In an intention to treat analysis all randomized subjects should be analyzed in this case all 262.The per-protocol analysis should be a secondary analysis.3.Both the OS and DFS analyses should include all deaths as events since these are not disease specific mortality, but overall survival.4. DFS should include both recurrence and death as events.Since probability of disease-free survival is being estimated, death can't be censored.5.For the primary analysis, DFS should start from randomization. 6. RMST graphs are not needed for OS since the curves should be the same as the KM graphs, but do not seem to be the same.7. The statistical analysis section does not include many of the analysis performed and the references for them.For example: a.The methods for the estimation of the HR are not specified.b.The methods for obtaining the 95%CI in line 154 c.The method for the p-value in line 155.d.Line 155, univariate analysis of OS at 3 year, what test was used?
Minor concerns: 1. Specify how median follow-up time was estimated.2. In line 154, should it be mean survival time instead of median? 3.In line 353, what is the unilateral statistical significance versus the alpha?4. In line 354, do you mean power instead of efficiency? 5. X-axis for OS KM should be time from randomization.
Thank you very much for your comments concerning our manuscript entitled "Effect of Intraoperative Denonvilliers' Fascia Preservation on Urogenital Function Protection and Oncological safety for Rectal Cancer" (Manuscript ID: NCOMMS-23-15046A).
Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research.We have studied the comments carefully and the followings are our responses to the reviewer point by point.
We also upload a revised version where the revisions were marked in yellow.

Responses to the reviewer's comments:
Reviewer #1: 1.The authors state that traditional TME requires partly resection of Denonvilliers' fascia, but in my view this is debatable and not generally acknowledged anymore.The rationale of performing this trial in the current era is not completely clear to me, because TME surgery is often individualized based on tumour location and extent since we have detailed information from preoperative MRI, aiming at an optimal balance between oncological safety and nerve preservation.Therefore, most surgeons will not resect Denonvilliers' fascia if the tumour is located on the dorsal or dorsolateral side, and this might even be safe if there is at least 2 mm margin on MRI to this fascia in anteriorly located tumours.The authors confirm that preserving Denonvilliers' fascia, if oncologically safe, gives the best outcomes.But in my opinion, this is common practice, at least in my country, and the trial does not provide any practice changing results.This discussion is especially relevant, since only a quarter of included patients had an anteriorly located tumour (Table 1).Therefore, the issue of preservation of Denonvilliers' fascia was only clinically relevant in a minor subgroup.
Response: Thanks for the extremely professional comment.We strongly agree that TME should be performed individualized based on tumor location and extent.However, this opinion has not been widely accepted, especially in China and other Asian countries.
In 2004, R.J. Heald described a holy plane for TME surgery and demonstrated that there was usually no surgical plane posterior to Denonvilliers' fascia (DVF).Thus, the optimal TME for rectal cancer was by dissection anterior to DVF and a U-shaped cut of DVF should be performed to avoid damage of bilateral neurovascular bundles (NVB) [1].Since Professor Heald is honored as "father of TME", seldom surgeons doubt with his opinion, and thus traditional TME with partly DVF resection was still routinely performed in many large-scale medical centers of China even when the tumor was located on the dorsal or dorsolateral side, or T1-2 anterior wall.When we presented the potential necessity and advantage of TME surgery with DVF preservation in large academic conferences previously, many colorectal surgeons doubted it.In fact, there are still researches and multicentre RCT in progress to investigate the necessity of TME surgery with routine partly resection of DVF [2][3][4].Thus, we believe the surgical approach of TME is still largely controversial.
In addition, although some surgeons, like you and us, agree that DVF should not be routinely resected in TME surgery, there is still no RCT so far to confirm the effect of DVF preservation on urogenital function preservation compared to partly resection in TME surgery.Thus, we believe that this RCT study is in urgent need to provide solid evidence on advantage of TME with DVF preservation and thus clarify the appropriate surgical ideas of TME.Although only a quarter of included patients had an anteriorly located tumor, this study also enrolled patients with T1-4N0-2M0 un-anterior tumor, and proved that TME with DVF preservation was not only oncological safe, but also of urogenital function advantage for all enrolled patients.Thus, we believed that the issue of DVF preservation was not only clinically relevant in a minor subgroup.We have added some of these statements which were marked in yellow in the Introduction Section of revised paper.Specially thanks for your professional comment.
[2] Zheng Z, Ye D, Wang X, Lu X, Huang Y, Chi P. Effect of partial preservation versus complete preservation of Denonvilliers' fascia on postoperative urogenital function in male patients with low rectal cancer (PREDICTION): protocol of a multicentre, prospective, randomised controlled clinical trial.BMJ Open.2022;12(4):e055355.
2. There is an important issue related to extrapolation of these results to other countries and patient populations.Western populations might be different due to obesity and different anatomy, and more application of neoadjuvant radiotherapy.
Response: Thanks for the professional comment.It is true that patients from different countries may differ from characteristics and anatomy.In this study, we also found that different patients presented different morphologies of either DVF or surgical landmark line.As presented below, the surgical videos revealed different morphology of DVF in Figure 1R.Some were thinner while some may be thicker.Some were gray while others may be light red.Also, Figure 2R presented the surgical landmark line located at the lowest level of peritoneal reflection.Some cases of this white line were thinner while some were thicker.Nevertheless, this surgical line existed persistently, and as we illustrated in the manuscript, regardless of either mobilization of the peritoneal reflection or different morphology of DVF, dissection below this marker line resulted in easily entry posterior to DVF.Thus, we believe that different anatomy will not restrict the extrapolation of the results in this trial.Although the mean BMI was normal in this study, there were also some cases of overweight (n=64) and obesity (n=6), while the procedure was performed smoothly regardless of the high BMI.Usually, for patients with high BMI, hanging the peritoneal reflection with suture (Figure 3R) or performing traction of the rectum with tieback (Figure 4R) will help better exposure of either pelvic cavity or the anterior wall of rectum, and thus make surgery easier to generalize.Also, as we discussed previously, the surgical marker line also existed persistently in patients with high BMI.Thus, DVF preservation with the guidance of this surgical marker line is also applicable in patients with obesity.We previously discussed with some colorectal surgeons from Japan, Korea and western countries, and they have also performed laparoscopic TME with DVF preservation for benign colorectal diseases or early-stage rectal cancers.Thus, we believe that this surgical procedure is generalizable in either China or other countries.We didn't enroll cases of neoadjuvant radiotherapy in this study because of the potential adverse effects of radiotherapy on urogenital function.In our clinical experience, cases with neoadjuvant radiotherapy, if needed, still could be performed TME with DVF preservation smoothly.Usually, for cases of neoadjuvant radiotherapy, electric hook was better than ultrasonic scalpel when performing pelvic dissection.
Nevertheless, as we discussed in the manuscript, we totally agree that further trial should be conducted to assess the feasibility and advantage of TME with DVF preservation for patients with neoadjuvant radiotherapy.
3. The two types of surgery are described as straight forward, but in reality it might sometimes be quite difficult to clearly identify Denonvilliers' fascia and to choose the plane either ventral or dorsal to this fascia.This is also due to high variability in appearance of Denonvilliers' fascia in different patients, as pointed out by the authors in the discussion.The authors describe video registration and photographs for quality control of the surgical interventions, but do not provide any results for assessment of videos or pictures.
Response: Thanks for the professional comment.It's such a good question that not only the professional reviewer, but also some experts doubt with this issue.In our very early stage of laparoscopic TME surgery, we usually dissected 1-1.5cm above peritoneal reflection when performing anterior dissection, and found that it was extremely difficult to dissect posterior to DVF.On the contrary, dissection at the lowest level of peritoneal reflection sometimes help enter the surgical plane posterior to DVF.We were confused why dissection on this procedure did not always lead to an appropriate surgical plane.
Then, through both cadaveric study and surgical videos review, we found that DVF began with a white thickened line at the lowest level of peritoneal reflection, and ended to the perineal body (Figure 5R).In other words, this white thickened line can be considered as surgical marker of DVF.Due to intraoperative traction and countertraction on the rectum, the fusion of the fascia was mobile and not always located at the lowest level of peritoneal reflection, which can explain why dissection at the lowest level of peritoneal reflection not always help enter posteriorly to DVF.On the contrary, the surgical marker of DVF is immobile, thus dissection below this marker line leads to entry posterior to DVF easily, while dissection above this marker line leads to entry anterior to DVF, regardless of mobilization of the peritoneal reflection (Figure 6R).With the help of this surgical line, identification of DVF and selection of two different procedures of anterior dissection has become feasible and practicable.
All intraoperative photographs of specific fields were reviewed and assessed to verify PAN protection, containing the anterior plane.All cases presented good PAN preservation.In the Exp-group, the DVF was well preserved, while DVF was partly resected as protocol in the Con-group.We have added some of these statements in the revised manuscript.4. Title: it is not clear from the title that this is an RCT.
Response: Thanks for the comment.According to the reviewer's suggestion, as well as due to the Nature Communications formatting instructions (no more than 15 words for the Title), we would like to revised the title to "Effect of Denonvilliers' Fascia Preservation on Male Urogenital Function and Oncological Safety (the PUF-01 Study)". 5. Discussion: This is too long and might benefit from more focus on the main findings of the study and comparing their results with urogenital function in other studies that included these outcomes, not necessarily looking at DVF preservation.The detailed anatomical discussion is probably something for a narrative review, but not as part of an original manuscript on an RCT.
Response: Thanks for the suggestion.We discussed anatomy in the present manuscript in order to clarify some misunderstandings of DVF and thus help readers better understand the meaning of this paper.Nevertheless, according to the reviewer's suggestion, we have simplified contents of Discussion Section.Also, we compared and discussed the incidence of urogenital dysfunction in this manuscript with other previous studies, which was marked in yellow in the first paragraph of Discussion Section.We wish that the revised Discussion Section would be more precise and of better quality.
6.The authors state that it would be interesting to also look at anterior T3 and T4 tumors, but this seems impossible or even unethical, as DVF is at least threatened or even involved in those patients.Please explain.
Response: Thanks for the professional comment.It's true that for anterior T3 and T4 tumors, it may not be suitable for dissection posterior to DVF due to threaten of tumor invasion.However, because DVF doesn't belong to mesorectum, it is not located within the "holy plane" and thus should not be resected even for T3 or T4a anterior tumor in theory.Whether preservation of DVF is suitable and possible for T3 or even T4a is controversial and unclear, so it may be further investigated in the future.Nevertheless, we absolutely agree that this clinical trial should be carefully assessed with ethical agreement before being conducted.
7. The fact that female patients were not included is not a limitation, because the DVF is not a clearly defined structure in women, and therefore the research question is not valid for this population.
Response: Thanks for the suggestion.We totally agree that the structure of DVF is more complicated and multiple-morphologic for female.In addition, the assessment method of sexual function for female is relatively insufficient.Thus, we did not enroll female rectal cancer patients in this study.According to the reviewer's suggestion, we have deleted the limitation description of female patients in the manuscript.In addition, we added the word "Male" in the Title of manuscript to follow the rules in the 'Sex and Gender Equity in Research-SAGER-guidelines' of the journal.

Special thanks for your professional comments and suggestions.
Reviewer #2: 1.The only thing a mis is information how many patients that were included at each participating center and whether all patients planned for a low anterior resection for curative intention were evaluated and included in the "group of eligible patients".
Response: Thanks very much for the reviewer's affirmation to our work.Please find attached the patient distribution among each participating center.Meanwhile, in most of the participating medical centers, patients planned for a low anterior resection for curative intention were routinely evaluated, then patients who met the inclusion criteria were included in the "group of eligible patients".The results in the text do not match the survival graphs and eTable5 presented.This is of major concern.The survival graphs sample size also don't match Table 1.For example, the log-rank p-values are different and the sample sizes are different for the stratified analysis.

Response:
We apologize for this mistake and confusion.First, we really appreciate the profession and rigor of the reviewer.These comments precisely hit the nail, thus greatly help us improve our manuscript and avoid stupid writing mistakes.In our previous manuscript, we did not include some cases of patients with APR surgery, thus some data were mixed by mistake and thus confusing.To avoid this, this time we invited two individual statisticians (Dr.Yang Shuo and Dr. Luo Hao from Department of Epidemiology and Biostatistics, School of Public Health, Sun Yat-Sen University) to analyze all data again and double check the revised version to be correct, containing Figure 2, Figure 3, Table 1, Table 2 and eTable 5. We are sorry for the statistic mistakes.
Also, in the Acknowledgments Section, we thank Dr. Yang Shuo and Dr. Luo Hao for their contribution of data re-analysis.
For the survival graphs (Figure 2   2. The analysis is stated as intention to treat, but it is per protocol.In an intention to treat analysis all randomized subjects should be analyzed in this case all 262.The perprotocol analysis should be a secondary analysis. Response: Thanks for the professional comment.It's true that for an ITT analysis, all randomized subjects should be analyzed.In this study, as shown in Figure 1, totally 262 patients were enrolled and randomly assigned to the Exp-group or Con-group (n=131 respectively).However, there were 9 cases in the Exp-group and 11 cases in the Congroup who accepted randomization but didn't finish laparoscopic rectal cancer resection (withdrew consent or had unresectable tumor intraoperatively).Thus, refer to previous studies [1,2], we used modified intention-to-treat analysis (mITT) for the remaining 122 cases in the Exp-group and 120 cases in the Con-group.We believe that mITT should be more objective than ITT to assess the survival outcome between the two groups in this case.To make it more precise, we have revised the description in the manuscript from ITT to mITT.
According to protocol, patients undergoing non-R0 resection or abdominal perineal resection (APR) were excluded for urogenital function assessment, thus 107 cases in the Exp-group and 100 cases in the Con-group were included for per-protocol analysis.
Thanks for the reviewer's kindly and professional suggestion.
References for this statement [1]  3. Both the OS and DFS analyses should include all deaths as events since these are not disease specific mortality, but overall survival.
Response: Thanks for the professional suggestion.We do agree that both the OS and DFS analyses should include all deaths as events.Thus, we have re-analyzed the data and redrawn the Figure 2 and 3.The revised results were marked in yellow in the revised manuscript and we wish that it would be more precise.
4. DFS should include both recurrence and death as events.Since probability of diseasefree survival is being estimated, death can't be censored.
Response: Consistent with the above reply, we agree that DFS should include both recurrence and death as events.We have changed death from censored to events and reanalyzed the data.Thanks for your kindly comment.
5. For the primary analysis, DFS should start from randomization.
Response: Thanks for reminding.We have corrected the description of DFS both in the text (Paragraph of Outcome measures in the Methods Section) and in the Figure 3, and also updated the data of DFS.
6. RMST graphs are not needed for OS since the curves should be the same as the KM graphs, but do not seem to be the same.
Response: Thanks for the comment.We have deleted the RMST graph from Figure 2 according to the suggestion, while the data of RMST were still preserved in the text.
Meanwhile, we double checked the RMST graphs and considered it to be the same as the KM graphs.The reason why it seemed that they were not the same may be ascribed to be different ordinate starting scale of Y-axis between the two graphs (The KM starts from 0% while the RMST starts from 50%).
7. The statistical analysis section does not include many of the analysis performed and the references for them.For example: a.The methods for the estimation of the HR are not specified.

Response:
We used Cox proportional hazard models to estimate hazard ratios (HRs) for quantifying the influence of various functional variables.The noninferiority margin for a hazard ratio (HR) of 1.34 was discussed and determined by Chinese Postoperative Urogenital Function (PUF) Research Collaboration Group and statistician, based on previous studies [1,2].We have added this statement in the Statistical analysis Section of revised manuscript, marked in yellow.
manuscript.These changes will not influence the content and framework of the paper.
And here we did not list the changes but marked in yellow in revised paper.We appreciate for Editors/Reviewers' warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.

2023.07.09
Reviewer #1: Remarks to the Author: I would like to thank the authors for thoroughly answering the comments and making the appropriate corrections to their manuscript.
Considering the revised version, I have some minor comments: 1.At the end of the introduction, the authors now have already summarized the main findings of their study.In my view, the last paragraph of the introduction should only include the aim of the study.
2. The first paragraph of the discussion should shortly summarize the main findings of the study.Subsequently the findings should be compared with relevant literature in the second paragraph.In the revised manuscript, the discussion now starts with some general statements about urogential function.So, I would propose to slightly rewrite the first part of the discussion.
3. The quality of the English writing should be improved with the help of a native English speaker.
Reviewer #3: Remarks to the Author: The revised paper and analysis have addressed most of the previous comments.The only remaining items that should be further clarified are: 1.I am still unclear why the log rank is different between the 3 and 5 year OS and DFS since the data is the same and the log rank test is used to compare the entire curve not a single time point.The analysis (Fig 2 and 3, and Table 2) should be based on all available follow-up as of the data lock for the comparison of OS and DFS.This way the text and Figures will match.The estimates of the probability of OS and DFS at specific time points can be reported along with their 95% CI even if the data does not end there.If patients are censored at 3 years that should be clarified in the methods.
In the interpretation, the authors should clarify that the logrank test and HR estimate are for the comparison of the OS and DFS curve, and not a specific test for the 3 year OS and DFS which the text seems to indicate.
2. All the statistical methods and references for the RMST and the log rank that were included in the response to reviewer's comments should also be included in the Methods section of the paper for clarity and reproducibility.Only some were included.

Figure 3R .
Figure 3R.Hanging the peritoneal reflection with suture to help exposure of the pelvic cavity

Figure 4R .
Figure 4R.Traction by tieback to help exposure of the pelvic cavity

Figure 5R .
Figure 5R.DVF originated from the lowest level of peritoneal reflection and formed as a thickened line.SV: seminal vesicle

Figure 6R .
Figure 6R.Two procedures for anterior wall dissection basing on the surgical marker line.A1-A3, dissection below marker line led to entry posterior to DVF and then DVF could be entirely preserved.B1-B3, dissection 1-1.5cm above marker line would directly enter the surgical plane anterior to DVF and DVF would be partly resected.

No
and 3), we drew the survival curve within 5 years in order to present more survival information, thus the log-rank p-values represented the statistic results of 5-year OS or DFS in Figure 2/3.While in the text, we presented the results of 3-year OS and DFS.That's why the log-rank p-values were different between the Figure and text description.Please find attached the 3-year OS and DFS graphs below, and if the reviewer consider it is better to use a same standard to describe the oncologic outcome, we would be willing to replace the Figure 2 and 3 to demonstrate the survival curve within 3 years.

Figure R1 Overall
Figure R1 Overall Survival for Exp-group vs Con-group at 3 years after surgery.
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